Extracellular arginine is required but the arginine transporter CAT3 (Slc7a3) is dispensable for mouse normal and malignant hematopoiesis

Amino acid-mediated metabolism is one of the key catabolic and anabolic processes involved in diverse cellular functions. However, the role of the semi-essential amino acid arginine in normal and malignant hematopoietic cell development is poorly understood. Here we report that a continuous supply of exogenous arginine is required for the maintenance/function of normal hematopoietic stem cells (HSCs). Surprisingly, knockout of Slc7a3 (CAT3), a major L-arginine transporter, does not affect HSCs in steady-state or under stress. Although Slc7a3 is highly expressed in naïve and activated CD8 T cells, neither T cell development nor activation/proliferation is impacted by Slc7a3 depletion. Furthermore, the Slc7a3 deletion does not attenuate leukemia development driven by Pten loss or the oncogenic Ptpn11E76K mutation. Arginine uptake assays reveal that L-arginine uptake is not disrupted in Slc7a3 knockout cells. These data suggest that extracellular arginine is critically important for HSCs, but CAT3 is dispensable for normal hematopoiesis and leukemogenesis.

Arginine is one of the most versatile amino acids with many metabolic and regulatory roles, serving as a proteogenic amino acid as well as a precursor for critical molecules, such as nitric oxide, creatine, polyamines, nucleotides, glutamate, etc., that have vital roles in cellular functions 1 . In addition, by binding to CASTOR (arginine sensor), arginine stimulates the activation of mTORC1 2-4 , a mast cell growth controller that integrates diverse environmental inputs to coordinate various anabolic and catabolic processes in cells. While some amino acids have been shown to be important for the maintenance or function of hematopoietic stem cells (HSCs) 5,6 , which are highly responsive to cell signaling and metabolic regulation during self-renewal and differentiation into mature blood cell lineages, little is known about the role of arginine for HSCs.
Arginine is a semi-essential amino acid since endogenous arginine produced from citrulline or breakdown of proteins is not sufficient for biosynthesis and signaling functions in certain types of cells or under conditions of high demand. Dietary arginine is thus indispensable to meeting metabolic demands 1 . Furthermore, arginine is implicated in cancer development and progression. Cancer cells consume significantly more arginine compared to their normal counterparts due to robust biosynthesis and the downregulation of argininosuccinate synthetase (ASS1), a rate-limiting enzyme in arginine synthesis, in a range of hematological and solid tumors 7 . As such, arginine starvation/degradation has emerged as a potential therapy for several types of cancers [8][9][10][11][12][13] . However, the role of arginine and its metabolites in various normal or malignant cells remains incompletely understood.
The availability of arginine for metabolic functions and other arginine-relevant biological processes is largely determined by the uptake of extracellular arginine via specialized transporters on the plasma membrane, cationic amino acid transporters (CATs) 14,15 . CAT3, encoded by Slc7a3 on the X chromosome, is primarily found in developing tissues of embryos, the adult thymus, and the brain [16][17][18] . This specific arginine transporter caught our attention because our previous gene expression profiling analyses revealed that the expression of Slc7a3 (CAT3), but not Slc7a1 (CAT1) or Slc7a2 (CAT2), was upregulated by 6.6 fold in the mitochondrial phosphatidylinositol phosphate phosphatase Ptpmt1 knockout HSCs, in which mitochondrial aerobic metabolism was suppressed 19,20 . This adaptive response of Slc7a3 expression in Ptpmt1 knockout HSCs raised the possibility that CAT3 may play

Results
Exogenous arginine is required but the arginine transporter CAT3 is dispensable for HSC maintenance and function. Our recent gene expression profiling showed that Slc7a3 (CAT3), a major arginine transporter, was greatly upregulated in Ptpmt1 knockout hematopoietic stem/progenitor cells in which mitochondrial aerobic metabolism was inhibited 20 . Quantitative RT-PCR (qRT-PCR) analyses verified that Slc7a3 expression levels were increased by ~ 17-fold in Ptpmt1 knockout Lin − Sca-1 + c-Kit + (LSK) cells (early progenitor cells enriched with HSCs) (Fig. 1A). This data suggests that arginine-mediated metabolism compensates for defective mitochondrial metabolism. Indeed, Slc7a3 expression increased by ~ eightfold when wild-type (WT) lineage negative (Lin − ) cells were treated with the mitochondrial inhibitor oligomycin (Fig. 1B). Given these observations, we sought to determine the potential role of arginine metabolism in hematopoiesis. We examined expression levels of Slc7a3 in hematopoietic cells in different developmental stages. Slc7a3 expression in HSCs, LSK cells, and myeloid progenitor LK (Lin − Sca-1c-Kit + ) cells was ~ 3 times higher than that in relatively mature Lin + cells (Fig. 1C). Furthermore, we cultured purified LSK cells in the absence of specific single amino acids and evaluated cell responses. After 7 days of culture, essentially all cells died in the wells without cysteine due to the essential role of this amino acid in the maintenance of cellular redox homeostasis. Interestingly, the deprivation of arginine from the culture medium also resulted in a robust detrimental effect (Fig. 1D). Arginine-deprived cells failed to proliferate and/or differentiate, and this effect was even greater than that caused by the deprivation of the essential amino acids valine, leucine, and isoleucine. These results suggest that a continuous supply of exogenous arginine is required for the maintenance and/or function of hematopoietic stem/progenitor cells.
We next determined the role of Slc7a3 in HSCs for hematopoietic regeneration in response to stress. We challenged Slc7a3 f/f /MX1-Cre + and Slc7a3 +/+ /MX1-Cre + mice with 5-fluorouracil (5-FU), a cell-cycle-dependent myelotoxic agent that kills cells in proliferation, including cycling hematopoietic stem and progenitor cells. White blood cell (WBC) counts dropped quickly in all 5-FU-treated mice. Subsequent hematopoietic recovery from quiescent HSCs occurred in Slc7a3 f/f /MX1-Cre + mice as efficiently as that in Slc7a3 +/+ /MX1-Cre + control littermates (Fig. 2D), suggesting that the deletion of Slc7a3 produces no deleterious effects on the regenerative capabilities of long-term HSCs that require robust energetic and metabolic support.
T cell development, activation, and proliferation are not disturbed in the absence of Slc7a3. Slc7a3 is highly expressed in the thymus 17 in addition to the brain 18 . Indeed, Slc7a3 has high expression levels in several CD8 T cell subsets in both mice and humans ( Figure S3). To determine the potential role of Slc7a3 in T cells, we examined T cells in the thymus and spleen in Slc7a3 f/f /Vav1-Cre + mice, but no defects in T cell development were observed. Percentages of double-positive (DP), double-negative (DN), CD4 + , and CD8 + T cells in the thymus ( Fig. 3A and B), and CD4 + and CD8 + T cells in the spleen (Fig. 3C) of the knockout mice were similar to those in control mice. Moreover, frequencies of naïve T cells (CD44 − CD62L + ), central memory T cells (CD44 + CD62L + ), and effector memory T cells (CD44 + CD62L − ) in both CD4 and CD8 T cell subpopulations were comparable in Slc7a3 knockout and control mice ( Fig. 3D and E), suggesting that the loss of Slc7a3 has no impact on T cell homeostasis. In addition, Slc7a3 knockout CD8 T cells could be efficiently activated, as evidenced by similar percentages of CD25 + and CD69 + cells derived from both knockout and control naive CD8 T cells (Fig. 3F). There was no difference in the proliferation of activated T cells between Slc7a3 knockout and WT control mice since frequencies of dividing cells (CFSE low ) in CD4 and CD8 T cell populations following anti-CD3/CD28 stimulation were similar in the two groups (Fig. 3G). Consistent with the activation and proliferation data, IFN-γ and granzyme B-producing cells derived from CD4 and CD8 populations were not affected by Slc7a3 depletion (Fig. 3H). These data demonstrate that Slc7a3 is dispensable for T cell development and activation.  www.nature.com/scientificreports/ reflected by enlarged spleens (Fig. 4B), increased WBC counts (Fig. 4C), and myeloid leukemic cells (Mac-1 + ) in the BM, spleen, and peripheral blood (PB) (Fig. 4D), was not mitigated in the double knockout mice. Furthermore, we examined the effect of Slc7a3 depletion on the development of the hematological malignancy induced by a gain-of-function mutation in Ptpn11 (Ptpn11 E76K/+ ) identified in juvenile myelomonocytic leukemia (JMML) 23 . Ptpn11 E76K/+ /Slc7a3 f/f /MX1-Cre + double mutant mice were generated by crossing Slc7a3 f/+ , MX1-Cre + , and inducible Ptpn11 E76K mutation knock-in mice (Ptpn11 E76Kneo/+ ) 24,25 . Both Ptpn11 E76K/+ /Slc7a3 Del double mutant and Ptpn11 E76K/+ single mutant mice uniformly developed a JMML-like myeloproliferative neoplasm, as demonstrated by the enlarged spleen (Fig. 5A) and markedly increased myeloid cells (Mac-1 + ) in the BM and spleen ( Fig. 5B and C). Altogether, these data suggest that the development and progression of hematopoietic malignancies are not dependent upon Slc7a3.
Slc7a3 is not required for mouse development. Slc7a3 has elevated expression levels in embryonic tissues 18 and is thought to play a major role during embryogenesis and fetal development 14,15 . In addition, Slc7a3 was found highly expressed in embryonic stem cells ( Figure S4). Our qRT-PCR analyses showed that Slc7a3 levels in mouse embryos at 8.5 days post coitum (dpc) were 5-10 times higher than those in adult tissues (Figure S5A). To determine if Slc7a3 is important for embryonic development, we generated Slc7a3 global knockout mice (Slc7a3 f/f /CMV-Cre + ) mice by crossing Slc7a3 f/+ mice with CMV-Cre + mice to delete Slc7a3 from the germline. These animals were born at the Mendelian ratio without any obvious defects. Their body weights were comparable to those of Slc7a3 +/+ /CMV-Cre + littermates during the 12-months follow-up ( Figure S5B). The knockout mice appeared normal, bred well, and had a regular lifespan. Moreover, histopathological examination revealed no abnormalities in adult tissues/organs in these knockout mice ( Figure S5C), suggesting that Slc7a3 is not required for mouse development or maintenance of tissue homeostasis. www.nature.com/scientificreports/ L-arginine uptake is not disrupted by CAT3 (Slc7a3) depletion. Since no significant defects were detected in either tissue specific or global Slc7a3 knockout mice, we examined arginine uptake in Slc7a3 knockout cells with 13 C-labeled L-arginine. Intracellular 13 C-L-arginine was detected and quantified by a Thermo TSQ   www.nature.com/scientificreports/ Quantis mass spectrometer coupled to a Thermo Vanquish UHPLC. To our surprise, Slc7a3 depleted cells isolated from Slc7a3 f/f /Vav1-Cre + and WT control littermates had similar arginine uptake efficiency (Fig. 6A), verifying that CAT3 is dispensable for arginine transportation. To determine if other arginine transporters might compensate for the loss of CAT3, we examined the expression of Slc7a1 (CAT1) and Slc7a2 (CAT2) in Slc7a3 knockout BM Lin − cells. No significant differences in Slc7a1 or Slc7a2 levels were detected between Slc7a3 knockout and WT control cells (Fig. 6B).

Discussion
Amino acids are essential as building blocks of proteins, energy sources, precursors of metabolites, and signaling molecules in the cell. Previous studies have demonstrated the important roles of essential amino acids valine and methionine in HSCs 5,6 . In the present study, we have unexpectedly found that exogenous arginine, a semiessential amino acid, is required for the maintenance and/or function of ex vivo cultured HSCs. Notably, the detrimental effect of arginine deprivation on HSCs was even greater than that caused by the starvation of essential amino acids such as valine 5 , and other important amino acids such as methionine 6 . The underlying mechanism is unclear. The important role of exogenous arginine for HSCs is also supported by the observation that Slc7a3 (CAT3), a major arginine transporter, was upregulated by ~ 17-fold in hematopoietic stem/progenitor cells that had defective mitochondrial metabolism 20 . Surprisingly, this transporter is dispensable for HSC maintenance and function. Hematopoietic cell development in steady-state or in response to hematopoietic stress was not affected in Slc7a3 knockout mice. Moreover, although Slc7a3 is highly expressed in naïve and activated CD8 T cells, neither T cell development nor activation/proliferation was impacted by Slc7a3 depletion. Arginine uptake assays revealed that arginine transportation was not disrupted in Slc7a3 knockout cells. Selective arginine depletion with recombinant arginine-degrading enzymes and arginine transportation inhibition has been exploited or proposed as a therapeutic strategy for the treatment of cancers due to the important role of arginine-mediated metabolism and signaling in cancer cells [8][9][10][11][12][13] . Our data presented in this report does not suggest that the arginine transporter CAT3 is a useful therapeutic target for controlling cancer cell access to arginine because Pten loss or an oncogenic Ptpn11 mutation-driven leukemia development is not impacted by Slc7a3 depletion. Arginine can be accumulated by a variety of amino acid transporter systems 14,15 . Three cationic amino acid transporters (CATs) are relatively selective for arginine but differ in their substrate affinities and sensitivities to trans-stimulation. Different CATs have distinct distribution patterns in various cell types. Their expression is highly regulatable at the levels of transcription, mRNA stability, translation, and subcellular localization 14,15 . Slc7a1 (CAT1) and Slc7a2 (CAT2) expression levels were not significantly changed in Slc7a3 (CAT3) knockout cells. It is possible that CAT1 and CAT2 were functionally upregulated to compensate arginine transportation in the absence of CAT3. Other Na + -independent cationic amino acid transport systems (system y + ) or heteromeric amino acid transporters could also compensate for the loss of CAT3 function. The specific amino acid transporter(s) that conduct(s) arginine transportation after Slc7a3 depletion remain(s) to be determined.

Materials and methods
Mouse studies. Slc7a3 f/+ mice were generated in this study by gene-targeting, in which Exon 4-9 were flanked by two Loxp sites through homologous recombination (Fig. S1A). Conditional Ptpn11 E76K mutation knock-in mice (Ptpn11 E76K-neo/+ ) were generated in our previous study 24 . MX1-Cre + , CMV-Cre + , and Pten f/+ mice in the C57BL6 background were obtained from The Jackson Laboratory. Vav1-Cre + mice were provided by Dr. www.nature.com/scientificreports/ Thomas Graf at Center for Genomic Regulation and ICREA, Spain. These mice were intercrossed to produce mice of various genotypes. Mice were kept under specific pathogen-free conditions in Division of Animal Resources, Emory University. Mice of the same age, sex, and genotype were randomly grouped for subsequent experiments (investigators were not blinded). All animal procedures complied with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Colony-forming unit assay. Freshly harvested BM cells (2 × 10 4 cells/ml) were seeded in 0.9% methylcellulose IMDM medium containing 15% fetal bovine serum (FBS), glutamine (10 -4 M), β-mercaptoethanol (3.3 × 10 -5 M), SCF (50 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), and erythropoietin (EPO, 3 units/ml). After 10 days of culture at 37 °C in a humidified 5% CO 2 incubator, hematopoietic colonies (CFU-GEMM, CFU-GM, and BFU-E) consisting of more than 50 cells were counted under an inverted microscope. The mean value of the triplicates from each mouse was used for final statistical analyses.

In-vitro culture of LSK cells in the medium lacking single amino acids. Sorted HSCs-enriched LSK cells (Lin
T cell activation and proliferation assay. Splenocytes freshly isolated from 6 to 8-week-old Slc7a3 f/f / Vav1-Cre + mice and control littermates were cultured in RPMI1640 medium supplemented with 10% FBS, 1% P/S, and hIL-2 (30 unit/ml) (1 × 10 6 cells/200 µl per well). These cells were stimulated with T-Activator Dynabeads (anti-CD3/CD28 antibodies) (2 µl/10 6 cells) for 24 h. The cells were collected and stained with CD4-PE-Cy7, CD8-BV605, CD69-APC/Cyanine7, and CD25-PE antibodies. Percentages of early activated T cell populations (CD25 + or CD69 + ) in CD4 + and CD8 + T cell populations were determined by FACS. For proliferation assays, freshly harvested splenocytes were loaded with CFSE (5 nM) for 10 min in a 37 °C 5% CO 2 incubator and washed three times with PBS before being seeded into culture plates (1 × 10 6 cells/200 µl). The cells were activated with T-Activator Dynabeads as described above and further cultured for 4 days. They were then collected and stained with CD4-PE-Cy7 and CD8-APC antibodies. Percentages of the cells with lower CFSE intensities were quantified. In addition, the harvested splenocytes were stimulated and cultured for four days as above and then treated with Cell Activation Cocktail (2 μL for each ml of cell suspension) and Golgistop (4 µl for every 6 ml of culture) for 4 h. The cells were collected and stained with CD4-PE-Cy7 and CD8-BV605 antibodies. They were then fixed and permeabilized by using a Cytofix/Cytoperm kit and stained with IFN-γ-APC, and Granzyme B-PE antibodies. Percentages of IFN-γ + and Granzyme B + cells were quantified by FACS.